1. Field of the Invention
The present invention relates to the field of antimicrobial peptides.
2. Related Art
Background
Granulysin, a protein expressed by human cytotoxic T lymphocytes (CTL) and natural killer cells, was discovered in 1987 by Drs. Krensky and Clayberger during a search for genes expressed late (3-5 days) after T cell activation. Granulysin has potent lytic activity against both Gram-negative and Gram-positive bacteria and fungi, including Mycobacterium tuberculosis (Mtb), Salmonella typhimurium, Escherichia coli, Staphlococcus aureus, Cryptococcus neoformus, Leishmania major, Leishmania monocytogenes, Candida albicans, Pseudomonas aeruginosa, and Vibrio cholera. 
Granulysin is a member of the saposin-like protein (SAPLIP) family that has been highly conserved for almost one billion years in various organisms. This family includes NK-lysin, a lytic molecule isolated from pig intestine; amoebapores, used by amoebae to kill bacterial invaders, and saposins. Naturally occurring granulysin is a 15 kD molecule which is processed intracellularly to a 9 kD protein. It consists of five alpha helices separated by short loop regions and four cysteine residues that form two intramolecular disulfide bonds. Similar to many other cationic peptides, granulysin lyses both microbes and mammalian cells.
Granulysin plays a central role in the human cellular immune response. T lymphocytes use granulysin to kill both extracellular and intracellular Mycobacterium tuberculosis, although the latter also requires perforin. Similarly, exocytosis of granulysin by CTL is required for killing of the fungus Cryptococcus as well as Plasmodium falciparum, the parasite that causes malaria. Granulysin expressing T cells were detected in cutaneous leprosy lesions at a six fold greater frequency in patients with the localized tuberculoid form as compared with the disseminated lepromatous form of the disease, strongly implicating granulysin in controlling the spread of leprosy. In contrast, perforin was expressed at similar frequencies in both types of leprosy. Although granulysin is apparently not lytic against either HIV or varicella-zoster virus, granulysin killed intracellular varicella virions and accelerated death of varicella infected cells. A study from a Japanese group reported that NK cells from cancer patients with a poor prognosis contained significantly less granulysin than NK cells from cancer patients with a good prognosis or from healthy donors. The levels of perforin were similar in cells from all three groups. In renal transplant recipients, elevated levels of granulysin in both PBL and renal biopsies are associated with acute rejection and poor graft outcome.
Stenger et al. U.S. Pat. No. 6,485,928 describes the use of granulysin as an antimicrobial agent. It further gives the sequence of human granulysin, which may also be found in GenBank Accession number AAN99767, defined herein as a “human granulysin sequence,”
(SEQ ID NO: 1)MATWALLLLA AMLLGNPGLE VSVSPKGKNT SGRESGFGWA IWMEGLVFSR LSPEYYDLAR AHLRDEEKSC PCLAQEGPQG DLLTKTQELG RDYRTCLTIV QKLKKMVDKP T /1/ QRSV SNAAT RVCRT /2/ GRSRW /3/ RDVCRNFMRR /4/ YQ SRVIQGLV /5/ AGETAQQICE DLRLCIPSTG PL
The H3 region is underlined and is shown again in Table 1, which also indicates alignments with “wild type,” or “parent,” i.e., non-mutated, sequences, of peptides further developed and tested as described below. As will be discussed below, the inserted numbers, /1/ through /5/, indicate the portions of granulysin from which the present peptides were originally obtained.
Bair U.S. Pat. No. 4,999,369 sets forth the nucleotide and predicted amino acid sequence of the “519” protein, referred to presently as granulysin. The amino acid sequences disclosed include two variants, having aa 16-145 and aa 16-172 of sequence id's no. 3 and 4 of the '928 patent. The '928 patent also discloses that the granulysin polypeptide may be modified with substitutions, insertions or deletions. These sequences may be used to modify or extend the peptides disclosed below.
Wang et al., “Bacterial and Tumoricidal Activities of Synthetic Peptides Derived from Granulysin,” J. Immunol. 165:1486-1490 (2000) discloses a number of synthetic peptides derived from granulysin. These peptides are derived from full-length granulysin and are intended to cover certain predicted alpha helices in granulysin. The authors termed their peptides G1 to G15. Certain substitutions were made in several of the peptides. C→S substitutions were made for each granulysin cysteine. These substitutions disrupted disulfide linkages in the peptide. The authors reported that both G8 (which does not contain C→S substitutions) and G9 (which does contain C→S substitutions) have the same bacterial lysing ability as full-length granulysin. Certain R→Q substitutions were also made in order to study the effects of positively charged residues on lysis. Positively charged residues were reported to be important for lytic activity.